Triptolide prodrugs

ABSTRACT

The invention provides compounds of formula (I): or a salt thereof. The invention also provides pharmaceutical compositions comprising a compound of formula I, processes for preparing compounds of formula I, intermediates useful for preparing compounds of formula I and therapeutic methods using the compounds of formula I.

RELATED APPLICATIONS

This application claims priority to United States ProvisionalApplication No. 61/176,249 that was filed on May 7, 2009.

BACKGROUND OF THE INVENTION

Pancreatic cancer is a particularly aggressive and devastating diseasewith a five-year survival rate of less than 5%. No effective drugtreatment is currently available which can effectively prolong patientsurvival. In 2006, over 35,000 new pancreatic cancer cases were reportedwith an almost equal number succumbing to the disease. Resistance toapoptosis has been investigated as a key factor in preventing responsein patients to therapies to treat pancreatic and other cancers.

Triptolide is a naturally occurring compound obtained from the plantTripterygium wilfordii. Triptolide is known to be useful in treatingautoimmune diseases, transplantation rejection (immunosuppression), andpossesses anticancer and anti-fertility effects as well as otherbiological effects (Qui and Kao, 2003, Drugs R. D. 4, 1-18). Triptolidehas strong antitumor effects against xenograft tumors (for example, Yanget al. Mol. Cancer. Ther, 2003, 2, 65-72). Triptolide is ananti-apoptotic agent with multiple cellular targets that are implicatedin cancer growth and metastasis. Triptolide inhibits NF-kB activation,induces bid cleavage, blocks induction of the survival gene p21WAF1/^(Cip1) (Wang et al. Journal of Molecular Medicine, 2006, 84,405-415) and inhibits the function of heat shock transcription factor 1(HSF1) thereby suppressing endogenous Hsp70 gene expression (Westerheideet al. 2006, Journal of Biological Chemistry, 281, 9616-9622).Triptolide also functions as a potent tumor angiogenesis inhibitor (Heet al. 2010, Int. Journal of Cancer, 126, 266-278).

Several mechanisms exist in living cells that protect against adverseconditions, including cancer cells. The synthesis of a family ofproteins referred to as heat-shock proteins (HSPs) is one suchprotective mechanism. Major HSPs include HSP90, HSP70, HSP60, HSP40 andsmaller HSPs. HSPs can be present in most intracellular compartments,with HSP70 being primarily located in cytosol.

Dysregulated expression of HSP70 is known to be associated with manydiseases including cancers. HSP70 is abundantly expressed in malignanttumors of various origins (For example: Hantschel et al. 2000, CellStress Chaperones, 5, 438-442), which render the tumor cells resistantto therapy and poor prognosis for the patient (Fuqua et al. 1994, BreastCancer Res, Treatment 32, 67-71). Heat shock protein 70 (Hsp70) is knownto be upregulated and over-expressed in pancreatic cancer cells ascompared to normal cells. Furthermore, HSP70 has a protective effect oncancer cells inhibiting apoptosis of the cells. Inhibition of HSP70 inpancreatic cancer cells has been shown to increase apoptic cell death ofthese cells (See for example Aghdassi et al., Cancer Research, 67(2) p.616-625 (2007)). Triptolide has been shown to inhibit pancreatic tumorgrowth and metastasis in mice. It was also shown that triptolide whenused in combination with ionization radiation its therapeutic effect inpancreatic cancer treatment is enhanced (Wang et al. Proc. Amer. Assoc.Cancer Res. 2006, 47, abstract #4720 and Wang et al. Clin. Cancer Res.2007, 13, 4891-4899). It is believed that the anticancer effectassociated with triptolide occurs as a result of reducing levels of theprotein HSP70 expressed in significant amounts by pancreatic cancercells as compared to normal pancreatic cells. Thus, triptolide therapieshave been of interest in the medical field for their potential treatmentof cancers that over-express HSP70, including pancreatic cancer. See forexample, Phillips et al., Cancer Research, 67(19), p. 9407-16 (2007).

There are, however, certain disadvantages associated with administeringtriptolide and different solutions to address these problems have beenexplored. One problem associated with native triptolide is that it isinsoluble in aqueous solution. Another problem associated with naturaltriptolide is poor bioavailability and toxic side effects. Triptolide,triptolide derivatives and certain prodrugs having improved solubilityand reduced toxicity are known. For example, Dai et al. U.S. Pat. No.6,548,537 describes triptolide prodrugs having increased solubility andreduced toxicity.

The phosphonoxymethyl moiety per se is known in the art for purposes offorming prodrug compounds of certain pharmaceutical compounds. Forexample, Krise et al., J. Med. Chem., 42, pp. 3094-3100 (1999) describespreparation of N-phosphonooxymethyl prodrugs of certain compounds toimprove water solubility.

Nevertheless, prodrugs must possess a number of properties in order tobe practically useful. For instance, desirable prodrugs should be stablefor formulation and administration. Additionally, once administered andpresent in the recipient's system, the prodrug must be successfullyactivated. Furthermore, both the prodrug and activated compound must becompatible with biological fluids, such as plasma and tissuehomogenates. Ultimately, the activated compound initially delivered inprodrug form must have its desired therapeutic or pharmaceutical effect.These and other factors can be difficult to achieve simultaneously, orcollectively balance, with certain types of compounds. Within thecontext of triptolide and triptolide prodrug compounds it has beendifficult achieve improved aqueous solubility, effective bioavailabilityfor oral dosage forms, faster in vivo release of triptolide, andrelatively reduced or lower toxicity in combination with significantinhibition of cancer cell growth. For example, see Chassaing et al.,Highly Water-Soluble Prodrugs of Anthelminthic Benzimidazole Carbamates:Synthesis, Pharmacodynamics and Pharmacokinetics, J. Med. Chem., 51(5),pp. 1111-1114 (2008).

Succinate prodrug forms of triptolide are known, but have beenassociated with certain disadvantages. See, for example, Harrousseau etal., Haematologica 2008, 93(s1), 14 Abstract 0038 and Kitzen et al.European Journal of Cancer 2009, 45, 1764-1772. Incomplete and variableconversion of the succinate prodrug of triptolide has been observed.

Thus, there exists a need in the medical and pharmaceutical fields forimproved therapeutics for treating cancers including aggressive solidtumor cancers, such as pancreatic cancer. There also exists a furtherneed for improved delivery or improved pharmacokinetic parameters orreduced toxicity of such therapeutics. There also exists a need forprodrug forms of triptolide that have improved solubility or that havefaster release of the active compound triptolide or that have a moretherapeutically effective release of the active compound triptolide orfor prodrug forms of triptolide with improved bioavailability.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a compound of the invention which isa compound of formula I:

wherein:

each R¹ is independently H, (C₁-C₆)alkyl, aryl(C₁-C₆)alkyl-,(C₃-C₆)cycloalkyl or aryl; and each R² is independently H, (C₁-C₆)alkyl,aryl(C₁-C₆)alkyl-, (C₃-C₆)cycloalkyl or aryl; or R¹ and R² together withthe atom to which they are attached form a (C₃-C₇)cycloalkyl; whereinany alkyl or cycloalkyl of R¹ or R² may be optionally substituted withone or more (e.g. 1, 2, 3, 4 or 5) groups selected from halo,(C₁-C₆)alkoxy and NR^(a)R^(b) and wherein any aryl of R¹ or R² may beoptionally substituted with one or more (e.g. 1, 2, 3, 4 or 5) groupsselected from halo, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, NR^(a)R^(b), nitro andcyano;

R^(a) and R^(b) are each independently selected from H, (C₁-C₆)alkyl,(C₃-C₆)cycloalkyl and aryl; or R^(a) and R^(b) together with thenitrogen to which they are attached form a pyrrolidino, piperidino,piperazino, azetidino, morpholino, or thiomorpholino;

n is 1, 2 or 3; and

each X is H;

or a salt thereof.

The invention also provides a pharmaceutical composition comprising acompound of formula I, or a pharmaceutically acceptable salt thereof, incombination with a pharmaceutically acceptable carrier.

The invention also provides a compound of formula I, or apharmaceutically acceptable salt thereof for use in medical therapy.

The invention also provides a method for treating cancer (e.g.pancreatic cancer, bile duct carcinoma, neuroblastoma, colon cancer,breast cancer, myeloma, gastric cancer, liver cancer, glioblastoma,ovarian cancer, colorectal cancer, non-Hodgkin lymphoma, lung cancer,prostate cancer, small-cell lung cancer, large cell lung cancer, kidneycancer, esophageal cancer, stomach cancer, cervical cancer or lymphomatumors) in a mammal (e.g. a human), comprising administering a compoundof formula I, or a pharmaceutically acceptable salt thereof, to themammal (e.g. a human).

The invention also provides a compound of formula I, or apharmaceutically acceptable salt thereof for use in the prophylactic ortherapeutic treatment of cancer (e.g. pancreatic cancer, bile ductcarcinoma, neuroblastoma, colon cancer, breast cancer, myeloma, gastriccancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer,non-Hodgkin lymphoma, lung cancer, prostate cancer, small-cell lungcancer, large cell lung cancer, kidney cancer, esophageal cancer,stomach cancer, cervical cancer or lymphoma tumors).

The invention also provides the use of a compound of formula I, or apharmaceutically acceptable salt thereof for the manufacture of amedicament for the treatment of cancer (e.g. pancreatic cancer, bileduct carcinoma, neuroblastoma, colon cancer, breast cancer, myeloma,gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectalcancer, non-Hodgkin lymphoma, lung cancer, prostate cancer, small-celllung cancer, large cell lung cancer, kidney cancer, esophageal cancer,stomach cancer, cervical cancer or lymphoma tumors.) in a mammal (e.g. ahuman).

The invention also provides a method for inhibiting cancer cell growthin an HSP70-expressing cancer (e.g. pancreatic cancer, neuroblastoma,breast cancer, colon cancer, gastric cancer, liver cancer orglioblastoma) in a mammal (e.g. a human) comprising administering aninhibitory effective amount of a compound of formula I, or apharmaceutically acceptable salt thereof, to the mammal (e.g. a human).

The invention also provides a compound of formula I, or apharmaceutically acceptable salt thereof for use in the prophylactic ortherapeutic inhibition of cancer cell growth in an HSP70-expressingcancer (e.g. pancreatic cancer, neuroblastoma, breast cancer, coloncancer, gastric cancer, liver cancer or glioblastoma).

The invention also provides the use of a compound of formula I, or apharmaceutically acceptable salt thereof for the manufacture of amedicament for the inhibition of cancer cell growth in anHSP70-expressing cancer (e.g. pancreatic cancer, neuroblastoma, breastcancer, colon cancer, gastric cancer, liver cancer or glioblastoma) in amammal (e.g. a human).

The invention also provides novel processes and novel intermediatesdisclosed herein that are useful for preparing compounds of formula I orsalts thereof, for example, those described in Schemes 1-2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a chemical reaction diagram for preparing thecompound 1.

FIG. 2 illustrates a chemical reaction diagram showing triptolide beingderivatized to compound 1 and the subsequent enzymatic cleavage-chemicalbreakdown of compound 1 to release triptolide.

FIG. 3 illustrates the in vitro enzymatic conversion of the triptolideprodrug (compound 1 to triptolide.

FIG. 4 illustrates the comparative effects of triptolide and thetriptolide prodrug (compound 1) on MiaPaca-2 cell viability in vitro at48 hours.

FIG. 5 illustrates the comparative effects of triptolide and thetriptolide prodrug (compound 1) upon Panc-1 cell viability in vitro atboth 24 hours and 48 hours.

FIG. 6 illustrates the comparative effects of triptolide and triptolideprodrug (compound 1) on S2VP10 cell viability in vitro at 24 hours and48 hours.

FIG. 7 illustrates tumor growth in a control group of mice with fivephotographs in situ and one photograph of tumors ex vivo.

FIG. 8 illustrates tumor growth in a triptolide group of mice with threephotographs in situ and one photograph of tumors ex vivo.

FIG. 9 illustrates tumor growth in a triptolide prodrug (compound 1)group of mice with four photographs in situ and one photograph of tumorsex vivo.

FIG. 10 is a photograph of the ex vivo tumor collection from the in vivoexperiment showing comparative tumor sizes for the control group,triptolide group and the triptolide prodrug (compound 1).

FIG. 11 illustrates comparative tumor weight (g) for the tumors of thecontrol group, triptolide group and the triptolide prodrug (compound 1)group of mice from the in vivo experiment.

FIG. 12 illustrates comparative tumor volume (cm³) for the tumors of thecontrol group, triptolide group and the triptolide prodrug (compound 1)group of mice from the in vivo experiment.

FIG. 13 illustrates survival analysis of the compound 1 treated mice andcontrol mice.

FIG. 14 illustrates survival analysis of the compound 1 treated mice andcontrol mice.

FIG. 15 illustrates tumor burden (volume and weight) for the compound 1triptolide and vehicle treated mice.

FIG. 16 illustrates tumor burden (volume and weight) for compound 1 andvehicle treated mice.

FIG. 17 illustrates tumor burden (volume and weight) for compound 1 andvehicle treated mice.

FIG. 18 illustrates tumor volume for compound and vehicle treated mice.

FIG. 19 illustrates cell viability (Neuroblastoma N2a and SKNSH) in thepresence of triptolide.

FIG. 20 illustrates Caspase 3 activity in the presence of triptolide.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “(C₁-C₆)alkyl” as used herein refers to alkyl groups havingfrom 1 to 6 carbon atoms which are straight or branched groups. Thisterm is exemplified by groups such as methyl, ethyl, n-propyl,iso-propyl, n-butyl, t-butyl, isobutyl, n-pentyl, neopentyl, andn-hexyl, and the like.

The term “(C₁-C₆)alkoxy” as used herein refers to the group(C₁-C₆)alkylO— wherein (C₁-C₆)alkyl is as defined herein. This term isexemplified by groups such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy, and thelike.

The term “(C₃-C₇)cycloalkyl” as used herein refers to a saturated orpartially unsaturated cyclic hydrocarbon ring system comprising 3 to 7carbon atoms. This term is exemplified by such groups as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexene, or cycloheptane, andthe like.

The term “aryl” as used herein refers to a phenyl radical or anortho-fused bicyclic carbocyclic radical having about nine to ten carbonring atoms in which at least one ring is aromatic. This term isexemplified by such groups phenyl, indanyl, indenyl, naphthyl,1,2-dihydronaphthyl and 1,2,3,4-tetrahydronaphthyl.

The term “aryl(C₁-C₆)alkyl-” as used herein refers to the grouparyl-(C₁-C₆)alkyl- wherein (C₁-C₆)alkyl and aryl are as defined herein.This term is exemplified by such groups as benzyl and phenethyl and thelike.

As used herein, the term “comprising” means the elements recited, ortheir equivalent in structure or function, plus any other element(s)which are not recited. The terms “having” and “including” are also to beconstrued as open ended unless the context suggests otherwise. Termssuch as “about,” “generally,” “substantially,” and the like are to beconstrued as modifying a term or value such that it is not an absolute,but does not read on the prior art. Such terms will be defined by thecircumstances and the terms that they modify are understood by those ofskill in the art. This includes at the very least the degree of expectedexperimental error, technique error, and instrument error for a giventechnique used to measure a value.

The phrases “therapeutically effective amount” and “pharmaceuticallyeffective amount” are used herein, for example, to mean an amountsufficient to reduce or inhibit in vivo cancerous cell growth uponadministration to a living mammal. The phrases are meant to refer to theamount determined to be required to produce the physiological effectintended and associated with the given active ingredient, as measuredaccording to established pharmacokinetic methods and techniques, for thegiven administration route.

The phrase “inhibitory effective amount” as used in association with theamount of active compound and composition of the invention is meant torefer, for example, to exhibited antitumor properties as demonstratedusing standard cell culture assay techniques.

As used herein, the term “prodrug” is meant to refer to a pharmaceuticalcompound that requires further metabolism (including but not limited tothe liver) before becoming biologically active.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms, forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase.

A salt of a compound of formula I can be useful as an intermediate forisolating or purifying a compound of formula I. Additionally,administration of a compound of formula I as a pharmaceuticallyacceptable acid or base salt may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts andinorganic salts.

The term “organic cation or inorganic cation” or “cationic organic orinorganic salt” include organic cations or inorganic cations (e.g. metalor amine salts) that are well known in the art and include cationicmoieties that can form an ionic association with the O moieties on thecompound and not significantly adversely affecting the desiredproperties of the prodrug for purposes of the invention. The term“pharmaceutically acceptable organic cations or inorganic cations” or“pharmaceutically acceptable cationic organic or inorganic salt” includethe “organic cations or inorganic cations” which are pharmaceuticallyacceptable for use in a mammal and are well known in the art.

Organic cations or inorganic cations include but are not limited tolithium, sodium, potassium, magnesium, calcium, barium, zinc, aluminiumand amine cations. Amine cations include but are not limited to cationsderived from ammonia, triethylamine, tromethamine (TRIS),triethanolamine, ethylenediamine, glucamine, N-methylglucamine, glycine,lysine, ornithine, arginine, ethanolamine, choline and the like. In oneembodiment, the amine cations are cations wherein X⁺ is of the formulaYH⁺ wherein Y is ammonia, triethylamine, tromethamine (TRIS),triethanolamine, ethylenediamine, glucamine, N-methylglucamine, glycine,lysine, ornithine, arginine, ethanolamine, choline and the like.

In one embodiment suitable cationic organic or inorganic salts that canbe used include cationic moieties that can form an ionic associationwith the O moieties on the compound and not significantly adverselyaffecting the desired properties of the prodrug for purposes of theinvention, e.g., increased solubility, stability, and rapid hydrolyticrelease of the active compound form. Preferably, X is selected from Li⁺,K⁺, or Na⁺. More preferably, X is Na⁺ thus forming the disodium salt.

Pharmaceutically acceptable salts can also include salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartrate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts. Salts, includingpharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion.

The invention includes both the free acid (e.g. —OP(O)(OH)₂), mono-salts(e.g. —OP(O)(OH)(O⁻X⁺)) and di-salts (e.g. —OP(O)O⁻X⁺)₂) of thecompounds of formula I. The acid and the salts may be purified by avariety of techniques well known in the art such as chromatography,followed by lyophilization or recrystallization.

It will be appreciated by those skilled in the art that a compound offormula I wherein X⁺ is an organic cation or inorganic cation can beconverted to a compound of formula I comprising one or more differentorganic or inorganic cations. Such a conversion can be accomplishedusing a variety of well known techniques and materials including but notlimited to ion exchange resins, ion exchange chromatography andselective crystallization.

A specific value for R¹ is H or (C₁-C₆)alkyl.

Another specific value for R¹ is H.

Another specific value for R¹ is (C₁-C₆)alkyl.

Another specific value for R¹ is methyl or ethyl.

A specific value for R² is H or (C₁-C₆)alkyl.

Another specific value R² is H.

A specific value for X⁺ is H.

Another specific value X⁺ is lithium, sodium, potassium, magnesium,calcium, barium, zinc or aluminium.

Another specific group of compounds of formula I are compounds whereinX⁺ is of the formula HY⁺ wherein Y is ammonia, triethylamine,tromethamine, triethanolamine, ethylenediamine, glucamine,N-methylglucamine, glycine, lysine, ornithine, arginine, ethanolamine orcholine.

Another specific value for X⁺ is Li⁺, K⁺ or Na⁺.

Another specific value for X⁺ is Na⁺.

A specific compound of formula I is 4-O-phosphonooxymethyltriptolidedisodium salt, 14-O-phosphonooxyethyltriptolide disodium salt or14-O-phosphonooxypropyltriptolide disodium salt, or a salt thereof.

A specific group of compounds of formula I are compounds formula Ia:

wherein X⁺ is a pharmaceutically acceptable organic cation or inorganiccation.

Another specific group of compounds of formula I are compounds formulaIa:

wherein X⁺ is a pharmaceutically acceptable cationic organic orinorganic salt.

Processes which can be used to prepare compounds of formula I andintermediates useful for preparing compounds of formula I are shown inScheme 1 and Scheme 2.

wherein Q is a protecting group such as benzyl or tert-butyl.

A compound of formula I can be prepared by removing one or moreprotecting groups from a compound of formula IA:

to provide the corresponding compound of formula I. Thus, theintermediate of formula IA is useful for preparing a compound of formulaI.

A compound of formula I can also prepared by converting the —SMe groupfrom a compound of formula IB:

to a —OP(O)(O⁻X⁺)₂ group to provide the corresponding compound offormula I. Thus, the intermediate of formula IB is useful for preparinga compound of formula I.

A compound of formula I can also be prepared by removing one or moreprotecting groups from a compound of formula IC:

to provide the corresponding compound of formula I. Thus, theintermediate of formula IC is useful for preparing a compound of formulaI.

A compound of formula I can also prepared by converting the —SMe groupfrom a compound of formula ID:

to a —OP(O)(O⁻X⁺)₂ group to provide the corresponding compound offormula I. Thus, the intermediate of formula ID is useful for preparinga compound of formula I.

Accordingly, the invention provides a method:

a) for preparing a compound of formula I comprising deprotecting acorresponding compound of formula IA bearing one or more protectinggroups to provide the compound of formula I.

b) for preparing a compound of formula I comprising converting the —SMegroup from a compound of formula IB to a —OP(O)(O⁻X⁺)₂ group to providethe compound of formula I.

c) for preparing a compound of formula I comprising deprotecting acorresponding compound of formula IC bearing one or more protectinggroups to provide the compound of formula I.

d) for preparing a compound of formula I comprising converting the —SMegroup from a compound of formula ID to a —OP(O)(O⁻X⁺)₂ group to providethe compound of formula I.

e) for preparing a salt of a compound of formula I comprising treating acorresponding compound of formula I with an acid (e.g. an organic acidor inorganic acid) or base (e.g. an alkali base or alkaline base) toprovide the salt of the compound of formula I.

f) for converting the a compound of formula I wherein one or more X⁺ isa cationic organic or inorganic salt to a compound of formula I whereinone or more X⁺ is a different cationic organic or inorganic salt.

The compound of the invention can be formulated into pharmaceuticalcompositions as well by combining together with a pharmaceuticallyacceptable carrier. Pharmaceutical compositions can be prepared inaccordance with well-known compounds and techniques readily available tothose skilled in the pharmaceutical field. For purposes of theinvention, the pharmaceutically acceptable carrier can be anyconventional and readily available biologically compatible or inertsubstance which is chemically compatible with the active pharmaceuticalingredient and does not significantly attenuate its intended therapeuticeffect upon formulation or delivery. Pharmaceutically acceptable saltscan be prepared using standard procedures and techniques well known inthe art.

The solid form of a compound of the invention can be a nanoparticle andthus formulated as a nanoparticle. Accordingly, the invention providesfor nanoparticles of a compound of formula I and compositions thatcomprise nanoparticles of a compound of formula I.

The triptolide prodrug compounds of the invention can be formulatedusing a variety of excipient formulations and prepared in various dosageforms as described below. The chemical properties and attributesassociated with the compounds of invention also can afford thepreparation of an oral solid dosage forms of the compounds of theinvention.

The compound of the invention can be formulated as pharmaceuticalcompositions and administered to a recipient in a variety of formssuitable for the desired particular administration route or system.Administration routes can include but are limited to oral routes,parenteral routes, intravenous routes (including intravenous routes bypump injection), intramuscular routes, topical routes including eyedrops, subcutaneous routes and mucosal routes. Compounds of theinvention can be administered systemically, e.g. orally, in combinationwith a pharmaceutically acceptable carrier such as an inert diluent orassimilable edible carrier. Thus the pharmaceutical compositioncomprising the compounds of the invention as the active ingredient canbe prepared in a variety of dosage forms. For example, the compositionscan be encapsulated in hard or soft capsules (e.g., gelatin orvegetable-derived capsular materials). The compositions can becompressed into ingestible or transmucosal tablet form, troches,capsules, elixirs, suspensions, syrups, wafers, suppositories and thelike. The amount of active ingredient can vary according to the specificdesired pharmaceutically effective dosage amount.

Tablets, troches, pills, capsules, and the like can contain additionalingredients such as binders (such as gum tragacanth, acacia, corn starchor gelatin); excipients such as dicalcium phosphate; disintegrants suchas corn starch, potato starch, alginic acid, and the like; lubricants(such as magnesium stearate) which can be used for tablet compressiontechniques, for example; sweeteners such as sucrose, fructose, lactoseor aspartame; and flavoring agents such as peppermint, wintergreen,cherry, and the like. Additional ingredients which may be included incompositions of the invention are mannitol, urea, dextranes, and lactosenon-reducing sugars.

When the dosage form is a capsule, it can contain a liquid carrierincluding polyethylene glycol, vegetable oil, etc. Other materials thatcan be used with certain dosage forms include gelatin, wax, shellac,sugar, and the like. Syrups or elixir forms can contain sucrose,fructose as sweeteners, methyl and propylparabens as preservatives, dyesand colorants, and flavoring agents.

When administered intravenously or intraperitoneally by infusion orinjection, solutions of the active ingredient and its salts can beprepared in, for example, water or saline optionally containing anon-toxic surfactant. Dispersions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils.Storage conditions may necessitate the inclusion of a preservative aswell.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Accordingly, the invention includes sterile preparation of a compound ofthe invention. The invention also includes non-sterile preparations of acompound of the invention.

Injectible or infusible pharmaceutical dosage forms can include sterileaqueous solutions or dispersions or sterile powders comprising theactive compounds of the invention prepared for extemporaneousformulation. Liquid carriers can include solvents or liquid dispersionmediums comprising water, ethanol, a polyol (e.g., glycerol, propyleneglycol, polyethylene glycols), and the like. Various agents can be addedto inhibit or prevent antimicrobial activity, such as parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Compounds and compositions of the invention can be administered as asingle dose or in multiple dose intervals. The dosage amount, dosageform, route of administration, and the particular formulationingredients can vary corresponding to the desired plasma concentrationand pharmacokinetics involved. A significant aspect of the invention isthat the particular compounds of the invention may afford an improvedand effective oral dosage form administration route by virtue of thecharacteristics and properties associated with the inventive compoundstructure and substituent location.

For topical administration, it will generally be desirable to administerthe compounds of the invention to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about3 to about 100 μg/kg of body weight per day (e.g. from about 6 to about96 μg/kg of body weight per day or from about 6 to about 48 μg/kg ofbody weight per day, or from about 6 to about 24 μg/kg of body weightper day, or from about 12 to about 24 μg/kg of body weight per day).

The compound is conveniently formulated in unit dosage form; forexample, containing from about 80 μg to about 8000 μg, conveniently fromabout 480 μg to about 7680 μg, conveniently from about 480 μg to about3840 μg, and conveniently from about 960 μg to about 1920 μg. In oneembodiment, the invention provides a composition comprising a compoundof the invention formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Compounds of the invention can also be administered in combination withother therapeutic agents, for example, other agents that are useful forthe treatment of cancer (e.g. pancreatic cancer, ovarian cancer,colorectal cancer, non-Hodgkin lymphoma, leukemia, acute and chronicmyelogenous leukemia, neuroblastoma, thyroid carcinoma, osteosarcoma,breast, prostate cancer, esophageal cancer, bladder cancer, gastriccarcinoma, urothelial cancer, glioblastoma multiforme, colon cancer,uterine cervical cancer, fibrosarcoma, squamous cell carcinoma, multiplemyeloma, cholangiocarcinoma, non-small cell lung cancer) as a radiationsensitizer for cancer cells, inflammatory diseases, rheumatic diseases,auto-immune diseases, polycystic kidney disease, nephritis.,transplantation graft survival (kidney, heart), pulmonary hypotension,lung inflammation, lung fibrosis, neuroprotection, cerebralischemia/reperfusion injury, Parkinsonism and corneal ulcers. Examplesof such agents include 5-fluorouracil, TRAIL (TNF-relatedapoptosis-inducing ligand), DR-4/5 activating antibodies,cyclophosphamide, hydroxydaunorubicin (doxorubicin), oncovin(vincristine), paclitaxel, doxetaxel, cisplatin, carboplatin, CPT-11,bortezimib and prednisone-prednisolone. Accordingly, in one embodimentthe invention also provides a composition comprising a compound offormula I, or a pharmaceutically acceptable salt thereof, at least oneother therapeutic agent, and a pharmaceutically acceptable diluent orcarrier. The invention also provides a kit comprising a compound offormula I, or a pharmaceutically acceptable salt thereof, at least oneother therapeutic agent, packaging material, and instructions foradministering the compound of formula I or the pharmaceuticallyacceptable salt thereof and the other therapeutic agent or agents to ananimal (e.g. mammal) to treat cancer (e.g. pancreatic cancer, ovariancancer, colorectal cancer, non-Hodgkin lymphoma, leukemia, acute andchronic myelogenous leukemia, neuroblastoma, thyroid carcinoma,osteosarcoma, breast, prostate cancer, esophageal cancer, bladdercancer, gastric carcinoma, urothelial cancer, glioblastoma multiforme,colon cancer, uterine cervical cancer, fibrosarcoma, squamous cellcarcinoma, multiple myeloma, cholangiocarcinoma, non-small cell lungcancer), an inflammatory disease, a rheumatic disease, an auto-immunedisease, a polycystic kidney disease, nephritis, transplantation graftsurvival (kidney, heart), pulmonary hypotension, lung inflammation, lungfibrosis, neuroprotection, cerebral ischemia/reperfusion injury,Parkinsonism, corneal ulcers or colitis. In another embodiment theinvention also provides a kit comprising a compound of formula I, or apharmaceutically acceptable salt thereof, at least one other therapeuticagent, packaging material, and instructions for administering thecompound of formula I or the pharmaceutically acceptable salt thereofand the other therapeutic agent or agents to an animal (e.g. mammal) tosensitize cancer cells, coat stents (drug elution), repair spinal cordrepair, or for use as in male and female contraception in animals. Thefollowing documents relate to triptolide in combination with othertherapeutic agents (1. Chen, Y. W. et al., Anticancer Drugs, 2010,21(5), 502-13. 2. Xu B. et al., Cancer Lett., 2010, 291(2), 200-208. 3.Borja-Cacho, D. et al., J. Gastrointest. Surg., 2010, 14(2), 252-60.Westfall S. D. et al., Chemotherapy, 2008, 54(1), 67-76. 4. Tang X. Y.et al., Postgrad. Med. J., 2007, 83(979), 338-43. 5. Panichakul T. etal., Anticancer Res. 2006, 26(1A), 259-65. 6. Pediatr. Blood Cancer,2008, 51(6):754-97. Matsui et al. Oncogene, 2008, 27, 4603-4614. 7.Chang et al., The Journal of Biological Chemistry, 2001 276, 2221-2227.8. Westfall et al., Chemotherapy, 2008, 54(1), 67-76. 9. Carter et al.,Blood, 2008, Vol. 111, No. 7, pp. 3742-3750. 10. Borja-Cacho et al., J.Gastrointest Surg., 2010, 14, 252-260. 11. Tang et al., PostgraduateMedical Journal 2007, 83, 338-343. 12. Cen et al., Anti-Cancer Drugs,2010, 21(5), 502-513. 13. Kapoor, Int. J. Mol. Med. 2008, 22(4), 489-96.14. Fidler et al., Molecular Cancer Therapeutics, 2003, 2, 855).

An important aspect of the invention is that compounds of the inventionafford desirable combinations of pharmacokinetic properties, physicalproperties and therapeutic advantages as compared to other triptolideprodrug forms. The triptolide prodrug compounds of the invention exhibitdesirable combination of attributes including chemical stability,enhanced solubility, and rapid metabolic release of the activetriptolide from the prodrug form. Collectively, these properties provideimproved therapeutic anticancer effects. Such effects include theeffective inhibition of pancreatic cancer cells by inhibiting theprotective effects afforded by HSP70 within cells and resistance toapoptosis and treatments.

The chemical pathway of the metabolic and enzymatic cleavage of thetriptolide prodrug of Example 1 is shown in FIG. 2. The starting nativecompound (non-prodrug form) triptolide has poor water solubilitycharacteristics. The prepared compound of Example 1 exhibits a highlevel of solubility. When subjected to enzymatic cleavage andmetabolism, the compound of Example 1 ultimately releases the activeform of the triptolide compound.

The compounds and compositions of the invention can be employed as amethod for treating solid tumor cancers in a mammal in need of suchtreatment comprising administering a pharmaceutically effective amountof a compound as described above as the active ingredient. As used inthe context of methods of treatment, the term “mammal” includes humans.

The compound and composition of the invention can be effective toinhibit in vitro and in vivo cancer cell growth of HSP70-expressingcancers. Examples of HSP70-expressing cancers include pancreatic cancer,breast cancer, lung cancer, neuronal cancer, leukemia, neuroblastoma,colon cancer, gastric cancer, liver cancer, and glioblastoma.

Accordingly, in one embodiment, the invention includes the inhibition ofa cancer cell population of cells exhibiting over-expression ofheat-shock protein HSP70 by the administration of a compound of formulaI. Of specific importance to the invention is the effective cellinhibition effect upon HSP70-expressing pancreatic cancer cells, such asMia-Paca, Panc-1 and S2VP10 cells. Accordingly, in another embodimentthe invention provides a method for treating an S2 cancer (e.g. anS2VP10 or S2013 cancer) in a mammal (e.g. a human), comprisingadministering a compound of formula I, or a pharmaceutically acceptablesalt thereof, to a mammal (e.g. a human).

In both in vitro and living mammalian systems, the enzyme alkalinephosphatase converts the compound of Example 1 into the activetriptolide form as demonstrated in the examples herein. The enzymatichydrolysis half life (t½) for the compound of Example 1 indicates arelatively rapid conversion rate and, consequently, faster release ofthe active therapeutic form of the compound.

Triptolide is used to treat a variety of diseases such as inflammatorydiseases. Triptolide has also been implicated as a therapeutic agent totreat a variety of diseases. These diseases include cancer (e.g.pancreatic cancer, bile duct carcinoma, neuroblastoma, colon cancer,breast cancer, myeloma, gastric cancer, liver cancer, glioblastoma,ovarian cancer, colorectal cancer, non-Hodgkin lymphoma, lung cancer,prostate cancer, small-cell lung cancer, large cell lung cancer, kidneycancer, esophageal cancer, stomach cancer, cervical cancer, lymphomatumors), autoimmune diseases, transplant rejection, polycystic kidneydisease, inflammatory diseases, asthma, rheumatoid arthritis, systemiclupus erythematosus and nephritis. Triptolide has also been discussed inthe coating of stents (drug elution), spinal cord repair, colitis, andcontraception in male and female animals. Accordingly, the inventionincludes but is not limited to the use of the compounds of formula I totreat diseases including cancer (e.g. pancreatic cancer, bile ductcarcinoma, neuroblastoma, colon cancer, breast cancer, myeloma, gastriccancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer,non-Hodgkin lymphoma, lung cancer, prostate cancer, small-cell lungcancer, large cell lung cancer, kidney cancer, esophageal cancer,stomach cancer, cervical cancer, lymphoma tumors), autoimmune diseases,transplant rejection, polycystic kidney disease, inflammatory diseases,asthma, rheumatoid arthritis, systemic lupus erythematosus andnephritis. Compounds of formula I can also be can also be used forcoating stents (drug elution), spinal cord repair, colitis, andcontraception in male and female mammals.

The following documents are directed to triptolide and cancer (1. AML:Carter et al., Blood, 2008, 111(7), 3742-3750. 2. Anaplastic thyroidcarcinoma: Mol. Pharmacol., 2009, 75(4), 812-9. 3. Bladder cancer: Yanget al., Mol. Cancer. Ther., 2003, 2(1), 65-72. 4. B16 Melanoma: Yang etal., Mol. Cancer. Ther. 2003, 2(1), 65-72. 5. Breast Cancer: Liang etal., Cancer Letters, 270(2), 2008, 337-341. Liu et al., Phytomedicine,2009, 16(11), 1006-1013. 6. Cervical Cancer Wang et al., J. Mol. Med.,2006, 84(5),405-15. 7. Cholangiocarcinoma: Tengchaisri et al., CancerLetters, 1998, 133(2), 169-175. 8. CML: Lou et al., Leukemia andLymphoma, 2004, 45, 373-376. 9. Colon: Tang et al., Postgraduate MedicalJournal 2007, 83, 338-343. 10. Esophageal cancer: Boult et al., B. J.Cancer, 2008, 89, 1985-92. 11. Fibrosarcoma: Kiviharju et al., ClinicalCancer Research, 2002, 8, 2666-2674. 12. Miyata et al., Biochem.Biophys. Res. Commun., 2005, 336(4), 1081-6. 13. Gastric Cancer: Jiang.,Oncogene, 2001, 20(55), 8009-18. 14. Yang et al., Mol. Cancer. Ther.2003, 2(1), 65-72. 15. Glioblastoma Multiforme Lin et al., J. Int. Med.Res., 2007, 35(4), 490-6. 16. Kapoor, Int. J. Mol. Med., 2008, 22(4),489-96. 17. Human Prostatic Epithelial Cells: Kiviharju et al., 2002.,Clinical Cancer Research, 8, 2666-2674. 18. Leukemias including AML:Carter et al., Blood, 2006, 108(2), 630-7. 19. Multiple myeloma: Yinjunet al., Leuk. Res. 2005 29(1), 99-105. 20. Neuroblastoma: Antonoff etal., Surgery, 2009, 146(2), 282-90.

21. non-Hodgkin lymphoma: Zhang et al., Acta Pharmacologica Sinica,2006, 27, 1438-1446. 22. Non-small cell lung cancer: Chang et al., TheJournal of Biological Chemistry, 276, 2221-2227. 23. Osteosarcoma: Wanget al., Pediatr. Blood Cancer. 2008, 51(6), 754-9. 24. Ovarian Cancer:Westfall et al., Chemotherapy, 2008, 54(1), 67-76. 25. PancreaticCancer: Wang et al., J. Mol. Med. 2006, 84(5), 405-15., Zhou et al.,World J., Gastroenterol, 2008, 14(10), 1504-1509., Wang et al. ClincalCancer Research 2007, 13, 4891., Phillips, Saluja et al., Cancer Res.,2007.Squamous cell carcinoma; Miyata et al., Biochem. Biophys. Res. Commun.,2005, 336(4), 1081-6. 26. Thyroid carcinoma: Zhu et al., Oncol Rep.,2009, 22(6),1397-401. 27. Uterine cervical carcinoma: Miyata et al.,Biochem. Biophys. Res. Commun., 2005, 336(4), 1081-6. 28. UrothelialCancer Matsui et al., Oncogene, (2008) 27, 4603-4614).

The following documents are directed to triptolide and diseases otherthan cancer (1. Multiple diseases: D Qui et al., Drug R & D, 2003, 4,1-16.

2. Organ transplantation: Chen, Leukemia and Lymphoma, 2001, 42,253-256.

3. Kidney transplant: Zhang et al., Journal of Ethnopharmacology, 2009,125(1), 141-46. 4. Transplantation graft survival (skin): Yang et al.Int. J. Immunophamac., 1992, 14, 963-969. 5. Graft-Versus-Host disease:Chen et al., Transplantation, 2000, 70, 1442-1447. 6. Inflammatory andautoimmune diseases: P. E. Lipsky et al., Seminars in Arthritis andRheumatism, 1997, 5, 713-723. 7. Autoimmune encephalomyelitis:Kizelsztein et al. Journal of Neuroimmunology, 2009, 217, 28-37. 8.Cerebral ischemia/reperfusion injury: Wei et al., Neural RegenerationResearch, 2007. 9. Colitis: Wei et al., Clin. Immunol. 2008, 129,211-218. 10. Contraception in males and females: Hikim et al., Journalof Andrology, 2000, 21, 431-437., Huynh et al., Journal of Andrology,2000, 21, 689-699., Wang et al., Asian Journal of Andrology, 1999, 1,121-125., Lue et al., Journal of Andrology, 1998, 19, 479-486. 11.Corneal ulcer: Lu et al. Investigative Ophthalmology and Visual Science.2006, 47, 3796-3800. 12. Lung inflammation: Krishna, et al., 2001, Am.J. Pathol., 2001, 158(3), 997-1004. 13. Nephritis: Tao et al., ArthritisRheum. 2008, 58(6), 1774-83. 14. Parkinsonism and neuroprotection: Zhouet al., Neurobiology of Disease, 2005, 18, 441-449. 15. Polycystickidney disease (PKD): Leuenroth et al., PNAS, 2007, 104, 4389-4394. 16.Spinal cord repair: Su et al., Glia 2010, 58, 901-915. 17. Stentcoating: Q. Luo 2005, Patent application 20050043788).

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLE 1 Synthesis of 14-O-phosphonooxymethyltriptolide disodium salt(compound 1)

To a solution of 14-O-phosphonooxymethyltriptolide dibenzyl ester (50mg, 0.08 mmol) in tetrahydrofuran (5 mL) was added palladium on carbon(10%, 10 mg). The mixture was stirred at room temperature under hydrogen(1 atm) for a period of 3 hours. The catalyst was removed by filtrationthrough CELITE™, and the filtrate was treated with a solution of sodiumcarbonate hydrate (8.9 mg in 3 mL water, 0.076 mmol). Thetetrahydrofuran was evaporated under reduced pressure and the residualwater solution was extracted with ether (3×3 mL). The aqueous layer wasevaporated to dryness and the resulting solid was dried overnight invacuo, washed with ether and again dried in vacuo to provide14-O-phosphonooxymethyltriptolide disodium salt (35 mg, 90% yield) as awhite powder. ¹H NMR (400 MHz, D₂O) δ 0.81 (d, 3H, J=6.8 Hz), 1.00 (d,3H, J=6.8 Hz), 1.03 (s, 3H), 1.35 (m, 1H), 1.50 (m, 1H), 2.00 (dd, 1H,J₁=14.7 and J₂=13.4 Hz), 2.08-2.61 (m, 4H), 2.85 (m, 1H), 3.63 (d, 1H,J=5.5 Hz), 3.81 (d, 1H, J=3.1 Hz), 3.86 (s, 1H), 4.12 (d, 1H, J=3.1 Hz),4.92 (m, 2H), 5.07 (m, 2H) ppm; ¹³C NMR (100 MHz, D₂O) δ 12.9, 16.0,16.3, 16.5, 22.3, 25.5, 28.9, 35.2, 39.8, 55.4, 56.1, 61.0, 61.5, 65.1,65.5, 71.9, 77.6, 91.7, 123.8, 164.2, 177.3 ppm; HRMS calculated for(C₂₁H₂₆O₁₀P) required m/z [M+1]⁺ 469.1264. Found m/z 469.1267.

Preparation of 14-O-phosphonooxymethyltriptolide dibenzyl ester

Step 1:

A solution of triptolide (100 mg, 0.29 mmol) in acetic acid (5 mL, 87.5mmol) and acetic anhydride (1 mL, 10.5 mmol) in DMSO (1.5 mL, 21.4 mmol)was prepared and stirred at room temperature for a period of 5 days toyield 14-O-methylthiomethyltriptolide intermediate. The reaction mixturewas then poured into water (100 mL) and neutralized with solid NaHCO₃,added in portions. The mixture was extracted with ethyl acetate (50mL×3), and the combined organic extract was dried over anhydrous sodiumsulfate and concentrated to furnish the product as an oil. Flash silicagel column chromatography (3:2 hexane/ethyl acetate) provided14-O-methylthiomethyltriptolide in 52% (60 mg) as a white foam. ¹H NMR(400 MHz, CDCl₃) δ 0.82 (d, 3H, J=6.8 Hz), 1.00 (d, 3H, J=6.8 Hz), 1.09(s, 3H), 1.20 (m, 1H), 1.59 (m, 1H), 1.93 (dd, 1H, J₁=14.7 and J₂=13.4Hz), 2.19 (s, 3H), 2.10-2.42 (m, 4H), 2.68 (m, 1H), 3.24 (d, 1H, J=5.5Hz), 3.51 (d, 1H, J=3.1 Hz), 3.67 (s, 1H), 3.79 (d, 1H, J=3.1 Hz), 4.68(m, 2H), 4.93 (d, 1H, J=11.8 Hz), 5.07 (d, 1H, J=11.8 Hz) ppm; ¹³C NMR(100 MHz, CDCl₃) δ 13.6, 14.8, 16.8, 17.0, 17.1, 23.4, 26.3, 29.5, 35.8,40.4, 54.5, 55.0, 58.0, 61.5, 63.9, 64.4, 69.9, 75.8, 76.7, 125.5,160.2, 173.2 ppm; HRMS calculated for (C₂₂H₂₈O₆SNa) required m/z [M+Na]⁺443.1505. Found m/z 443.1507.

Step 2:

A solution of 14-O-methylthiomethyltriptolide (50 mg, 0.12 mmol) in drymethylene chloride (2 mL) under an N₂ atmosphere was combined withpowdered activated 4 Å molecular sieves (50 mg), followed by theaddition of a mixture of dibenzylphosphate (40 mg, 0.14 mmol) andN-iodosuccinimide (32 mg, 0.14 mmol) in tetrahydrofuran (2 mL). Thereaction mixture was stirred at room temperature for a period of 5hours, filtered, and diluted with methylene chloride (20 mL). Theresulting solution was washed with a solution of sodium thiosulfate (2mL, 1M solution), a saturated solution of sodium bicarbonate, brine,dried over a sodium sulfate, filtered, and concentrated in vacuo. Theoily residue was purified by silica gel flash chromatography (1:2hexane/ethyl acetate) to give 14-O-phosphonooxymethyltriptolide dibenzylester (62 mg, 80% yield) as a white foam. ¹H NMR (400 MHz, CDCl₃) δ 0.72(d, 3H, J=6.8 Hz), 0.89 (d, 3H, J=6.8 Hz), 1.05 (s, 3H), 1.27 (m, 1H),1.48 (m, 1H), 1.82 (dd, 1H, J₁=14.7 and J₂=13.4 Hz), 2.03-2.35 (m, 4H),2.64 (m, 1H), 3.14 (d, 1H, J=5.5 Hz), 3.46 (d, 1H, J=3.1 Hz), 3.65 (s,1H), 3.76 (d, 1H, J=3.1 Hz), 4.65 (m, 2H), 5.02 (m, 4H), 5.27 (m, 1H),5.47 (m, 1H), 7.34 (m, 10H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 13.6, 16.8,17.0, 23.3, 26.2, 29.62, 29.67, 35.7, 40.3, 54.7, 55.2, 59.3, 61.1,63.6, 64.0, 69.36, 69.39, 69.42, 69.45, 69.9, 78.2, 92.9, 93.0, 125.5,127.9, 128.0, 128.6, 135.5, 135.6, 160.1, 173.2 ppm; HRMS calculated for(C₃₅H₃₉O₁₀PNa) required m/z [M+Na]⁺ 673.2179. Found m/z 673.2176.

EXAMPLE 2 Synthesis of 14-O-phosphonooxymethyltriptolide disodium salt(compound 1)

To a solution containing 14-O-methylthiomethyltriptolide (50 mg, 0.12mmol), phosphoric acid (82 mg, 0.84 mmol), and molecular sieves (4 Å,0.45 g) in THF (10 mL) at 0° C. was added N-iodosuccinimide (41 mg, 0.18mmol), and the mixture was stirred at room temperature for 1 h. Thereaction mixture was filtered through Celite, and the solids were washedwith THF. The filtrate was treated with 1 M Na₂S₂O₃ until it wascolorless and the filtrate was treated with a solution of sodiumcarbonate (13 mg in 3 mL water, 0.12 mmol). The filtrate was evaporatedunder reduced pressure and the residual water solution was extractedwith ether (3×3 mL). The aqueous layer was evaporated to dryness and theresulting residue was purified by chromatography (C18), eluting with agradient of 0-100% methanol in water to give14-O-phosphonooxymethyltriptolide disodium salt (43 mg, 70% yield) as acolorless powder.

Preparation of 14-O-methylthiomethyltriptolide

To a solution of triptolide (100 mg, 0.28 mmol) and methyl sulfide (0.16mL, 2.24 mmol) in acetonitrile (10 mL) at 0° C. was added benzoylperoxide (0.27 g, 1.12 mmol) in four equal portions over 20 min, andthen the mixture was stirred at 0° C. for 1 h and thereafter at roomtemperature for 1 h. The mixture was diluted with ethyl acetate andwashed with 10% Na₂CO₃ and then brine. The organic phase was dried overMgSO₄, filtered, and evaporated. The residue was purified by silica gelflash chromatography (1:1 hexane/ethyl acetate) to furnish14-O-methylthiomethyltriptolide (63 mg, 54% yield) as a colorlesspowder.

EXAMPLE 3 Synthesis of 14-O-Phosphonooxyethyltriptolide disodium salt

To a solution containing 14-O-methylthioethyltriptolide (52 mg, 0.12mmol), phosphoric acid (82 mg, 0.84 mmol), and molecular sieves (4 Å,0.45 g) in THF (10 mL) at 0° C. was added N-iodosuccinimide (41 mg, 0.18mmol), and the mixture was stirred at room temperature for 1 h. Thereaction mixture was filtered through Celite, and the solids were washedwith THF. The filtrate was treated with 1 M Na₂S₂O₃ until it wascolorless and the filtrate was treated with a solution of sodiumcarbonate (13 mg in 3 mL water, 0.12 mmol). The filtrate was evaporatedunder reduced pressure and the residual water solution was extractedwith ether (3×3 mL). The aqueous layer was evaporated to dryness and theresulting residue was purified by chromatography (C18), eluting with agradient of 0-100% methanol in water to give14-O-phosphonooxyethyltriptolide disodium salt (46 mg, 72% yield) as acolorless powder. ¹H NMR (400 MHz, D₂O) δ 0.68 (d, 3H, J=6.8 Hz), 0.70(d, 3H, J=6.8 Hz), 1.03 (s, 3H), 1.21 (m, 1H), 1.57 (d, 3H, J=5.3 Hz),1.58 (m, 1H), 1.94 (dd, 1H, J₁=14.7 and J₂=13.4 Hz), 2.08-2.61 (m, 4H),2.62 (m, 1H), 3.27 (d, 1H, J=5.5 Hz), 3.45 (d, 1H, J=3.1 Hz), 3.72 (d,1H, J=3.1 Hz), 3.79 (s, 1H), 4.63 (m, 2H), 6.43 (q, 1H, J=5.3 Hz) ppm;¹³C NMR (100 MHz, D₂O) δ 13.5, 16.9, 17.0, 17.1, 21.4, 23.5, 26.8, 29.5,35.9, 40.3, 54.0, 55.1, 59.4, 61.2, 63.6, 64.2, 69.8, 75.8, 76.5, 91.6,125.6, 164.2, 177.2 ppm; HRMS calculated for (C₂₂H₂₈O₁₀P) required m/z[M+1]⁺ 483.1137. Found m/z 483.1134.

Preparation of 14-O-methylthioethyltriptolide

To a solution of triptolide (100 mg, 0.28 mmol) and ethyl sulfide (0.24mL, 2.24 mmol) in acetonitrile (10 mL) at 0° C. was added benzoylperoxide (0.27 g, 1.12 mmol) in four equal portions over 20 min, andthen mixture was stirred at 0° C. for 1 h and then at room temperaturefor 1 h. The mixture was diluted with ethyl acetate and washed with 10%Na₂CO₃ and then brine. The organic phase was dried over MgSO₄, filtered,and evaporated. The residue was purified by silica gel flashchromatography (1:1 hexane/ethyl acetate) to give14-O-methylthioethyltriptolide (60 mg, 50% yield) as a colorless powder.¹H NMR (400 MHz, CDCl₃) δ 0.68 (d, 3H, J=6.8 Hz), 0.70 (d, 3H, J=6.8Hz), 1.04 (s, 3H), 1.20 (m, 1H), 1.57 (d, 3H, J=5.3 Hz), 1.59 (m, 1H),1.88 (dd, 1H, J₁=14.7 and J₂=13.4 Hz), 2.19 (s, 3H), 2.06-2.27 (m, 4H),2.62 (m, 1H), 3.24 (d, 1H, J=5.5 Hz), 3.42 (d, 1H, J=3.1 Hz), 3.70 (d,1H, J=3.1 Hz), 3.73 (s, 1H), 4.61 (m, 2H), 5.02 (q, 1H, J=5.3 Hz) ppm;¹³C NMR (100 MHz, CDCl₃) δ 13.6, 14.8, 16.9, 17.0, 17.1, 21.0, 23.5,26.4, 29.6, 35.8, 40.5, 54.0, 55.2, 59.4, 61.3, 63.7, 64.2, 69.9, 75.8,76.7, 125.6, 160.2, 173.2 ppm; HRMS calculated for (C₂₃H₃₀O₆SNa)required m/z [M+Na]⁺ 457.1763. Found m/z 457.1765.

EXAMPLE 4 Synthesis of 14-O-Phosphonooxypropyltriptolide disodium salt

To a solution containing 14-O-methylthiopropyltriptolide (54 mg, 0.12mmol), phosphoric acid (82 mg, 0.84 mmol), and molecular sieves (4 Å,0.45 g) in THF (10 mL) at 0° C. was added N-iodosuccinimide (41 mg, 0.18mmol), and the mixture was stirred at room temperature for 1 h. Thereaction mixture was filtered through Celite, and the solids were washedwith THF. The filtrate was treated with 1 M Na₂S₂O₃ until it wascolorless and the filtrate was treated with a solution of sodiumcarbonate (13 mg in 3 mL water, 0.12 mmol). The filtrate was evaporatedunder reduced pressure and the residual water solution was extractedwith ether (3×3 mL). The aqueous layer was evaporated to dryness and theresulting residue was purified by chromatography (C18), eluting with agradient of 0-100% methanol in water to provide14-O-phosphonooxypropyltriptolide disodium salt (43 mg, 65% yield) as acolorless powder. ¹H NMR (400 MHz, D₂O) δ 0.66 (d, 3H, J=6.8 Hz), 0.68(d, 3H, J=6.8 Hz), 0.99 (t, 3H, J=5.3 Hz), 1.03 (s, 3H), 1.20 (m, 1H),1.53 (m, 1H), 1.90 (dd, 1H, J₁=14.7 and J₂=13.4 Hz), 2.04-2.66 (m, 4H),2.65 (m, 3H), 3.27 (d, 1H, J=5.5 Hz), 3.49 (d, 1H, J=3.1 Hz), 3.71 (d,1H, J=3.1 Hz), 3.78 (s, 1H), 4.69 (m, 2H), 6.31 (q, 1H, J=5.3 Hz) ppm;¹³C NMR (100 MHz, D₂O) δ 7.55, 13.5, 16.2, 16.9, 17.2, 20.8, 23.2, 26.1,28.4, 34.7, 38.5, 54.1, 55.0, 59.0, 61.3, 62.5, 63.9, 68.5, 75.4, 76.4,91.9, 125.7, 160.1, 174.5 ppm; HRMS calculated for (C₂₃H₂₉O₁₀P) requiredm/z [M+1]⁺ 497.1294. Found m/z 497.1292

Preparation of 14-O-methylthiopropyltriptolide

To a solution of triptolide (100 mg, 0.28 mmol) and propyl sulfide (0.32mL, 2.24 mmol) in acetonitrile (10 mL) at 0° C. was added benzoylperoxide (0.27 g, 1.12 mmol) in four equal portions over 20 min, and themixture was stirred at 0° C. for 1 h and then at room temperature for 1h. The mixture was diluted with ethyl acetate and washed with 10% Na₂CO₃and then brine. The organic phase was dried over MgSO₄, filtered, andevaporated. The residue was purified by silica gel flash chromatography(1:1 hexane/ethyl acetate) to give 14-O-methylthiopropyltriptolide (60mg, 48% yield) as a colorless powder. ¹H NMR (400 MHz, CDCl₃) δ 0.65 (d,3H, J=6.8 Hz), 0.67 (d, 3H, J=6.8 Hz), 0.99 (t, 3H, J=5.3 Hz), 1.01 (s,3H), 1.20 (m, 1H), 1.59 (m, 1H), 1.88 (dd, 1H, J₁=14.7 and J₂=13.4 Hz),2.18 (s, 3H), 2.01-2.26 (m, 4H), 2.62 (m, 3H), 3.24 (d, 1H, J=5.5 Hz),3.42 (d, 1H, J=3.1 Hz), 3.70 (d, 1H, J=3.1 Hz), 3.73 (s, 1H), 4.61 (m,2H), 5.03 (q, 1H, J=5.3 Hz) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 7.68, 13.5,14.6, 16.2, 17.0, 17.2, 21.4, 23.2, 26.1, 28.9, 34.7, 39.5, 54.1, 55.6,59.0, 61.3, 63.5, 64.0, 69.5, 75.1, 76.4, 125.1, 160.9, 173.5 ppm; HRMScalculated for (C₂₄H₃₂O₆SNa) required m/z [M+Na]⁺ 471.1920. Found m/z471.1918.

EXAMPLE 5 Chemical Properties of the Compounds of Formula I

The chemical properties of the compounds of the invention wereevaluated. The aqueous solubility and chemical stability of the wasmeasured. Using a solution of the compound of Example 1 with pH adjustedto 7.4, solubility at room temperature was determined to be 61.4 mg/mL.Stability of the compounds of the invention were evaluated in Trisbuffer (pH 7.4) and Borate buffer (pH 7.4) solutions at roomtemperature. After a period of 1 (one) month, no degradation of thecompound of Example 1 was observed. The results are summarized in thefollowing table.

TABLE 1 Physiochemical Properties of Compounds of Formula I ChemicalChemical Enzymatic Solubility Stability Stability Hydrolysis (mg/mL)(t½) (t½), (t½), Tris Tris Borate Alkaline buffer, buffer, buffer,Phosphatase, Compound room temp room temp room temp. 37° C. Compound of61.4 * * 2 min. Example 1/2 Compound of >50 * * 9 min. Example 3Compound of >50 * * 17 min.  Example 4 * No degradation observed afterone month.

EXAMPLE 6 In Vitro Enzymatic Conversion Compound 1

Compound 1 is converted into the active tritpolide form by action of theenzyme alkaline phosphatase. An in vitro experiment to study thebioconversion of the tritpolide prodrug compound of the invention wasperformed. In vitro bioconversion was simulated using alkalinephosphatase (from bovine intestinal mucosa, Type VII-S available fromSigma-Aldrich (St. Louis, Mo.) in glycine buffer (pH 9.8).

Alkaline phosphatases are a group of enzymes found primarily in theliver (isoenzyme ALP-1) and bone (isoenzyme ALP-2), with small amountsbeing produced by the cellular lining of the small intestine (isoenzymeALP-3), placenta and kidneys. Alkaline phosphatases split offphosphorous to create an alkaline pH. Other enzymes in addition toalkaline phosphatase may contribute to in vivo hydrolysis as well.

From FIG. 3, it can be seen that the decreasing amounts of thetriptolide prodrug form were coincident with the proportionateincreasing amounts of active form released triptolide. Furthermore, itwas observed that the conversion occurred over a relatively short timeperiod, with the majority of conversion taking place over the initialtime period of 10 minutes.

The first order degradation constant was calculated by fitting theremaining concentration versus the incubation time. The degradationhalf-life (t ½) of compound 1 was determined to be 2 minutes.

EXAMPLE 7 Comparative In Vitro Cell Viability Study

An experiment was conducted to evaluate comparative in vitro cellviability using triptolide, the prodrug of triptolide (compound 1), andcontrol (without triptolide or the prodrug form). Cell viability wasdetermined using the Dojindo Cell Counting Kit-8 (available from DojindoLaboratories, Rockville, Md.). Pancreatic cancer cells were seeded intoa 96 well plate at 2×10³ cells per well and allowed to adhere overnight.The cells were then treated with the prodrug triptolide (compound 1), ofthe invention and native triptolide at various concentrations forperiods of 24 hours and 48 hours. Tetrazolium substrate (10 μl) wasadded to each well of the plate. The plates were incubated at 37° C. for1 hour, after which absorbance at 460 nm was measured. Each experimentwas performed in triplicate and repeated three independent times.

The effect of the compound 1 and triptolide on the viability ofpancreatic cancer cells was observed following incubation in mediumcontaining the prodrug tritpolide at concentrations ranging from 50 to200 nmol/L at 24 hours and 48 hours. The data was collected andconverted into the charts of FIG. 4 (Cell Viability Mia-Paca at 48hours), FIG. 5 (Cell Viability Panc-1 at 24 and 48 hours) and FIG. 6(Cell Viability S2VP10 at 24 and 48 hours).

As can be seen from each of the above data and figures, the presence ofthe triptolide prodrug and alkaline phosphatase and native formtriptolide significantly reduced pancreatic cell viability in vitro in atime- and dose-dependent manner

EXAMPLE 8 Comparative In Vivo Study of Compound 1 on Mice

Thirty nude mice of the strain athymic nu/nu were obtained from NationalCancer Institute (NCl) (Rockville, Md.) and kept in a RAR facility. Themice were anaesthetized in accordance with the recommendations of theRAR facility using ketamine 75-200 mg/kg and xylazine 4-8 mg/kg. A smallincision 3 mm was made on the left side of the abdominal wall and thespleen was pulled out with forceps far enough to expose and access thepancreas. MiaPaca-2 cell medium was prepared and kept on ice untildelivery into the mice. Into each of the mice, 1 million MiaPaca-2 cellssuspended in MATRIGEL™ (available from Becton-Dickinson Corporation,Franklin Lakes, N.J.) was injected into the tail of the pancreas(identified by its anatomical attachment with the spleen) using aHamilton syringe. Following delivery of the cells, the syringe was heldsteady for an additional 5-10 seconds to permit the MATRIGEL™ to set.The spleen was replaced back into the abdominal cavity, and theabdominal wall was closed by vicryl suture in a continuous manner. Theskin was apposed and closed using wound clips. The mice were thentransferred to a heating pad until fully recovered before being returnedto the cage. Post-operative pain medication (buprenonorphine 0.1 mg/kg)was administered intraperitoneally immediately after full recovery fromanaesthesia to prevent respiratory depression and then administeredevery 12 hours for 2 days. The wound clips were removed from the miceafter 7 days of surgery.

The mice were then randomized into 3 groups, each group having 10 mice.The groups were as follows: control group, triptolide group and thetriptolide prodrug (compound 1) group. The control group consisted ofmice that were injected intraperitoneally with vehicle DMSO.

The triptolide group subjects were injected with 0.2 mg/kg of triptolidedissolved in DMSO and diluted with phosphate buffered saline to a volumeof 100 μl, the intraperitoneal injections being daily over a period of60 days. The compound 1 subjects were intraperitoneally injected dailyfor 60 days with 0.28 mg/kg of the compound dissolved in phosphatebuffered saline diluted to a volume of 100 μl.

The mice were euthanized under anaesthesia at the conclusion of the 60day treatments. Samples were collected (blood, lung, spleen, liver,kidneys and tumor tissue), and tumor volume and weight were measured andcompared among the different groups. Observations were made on theloco-regional growth and cancer growth.

FIG. 8 (photographs from the control group), FIG. 9 (photographs of thetriptolide group) and FIG. 10 (photographs from the triptolide prodrugof Example 1 group), are a collection of photographs showing final tumorgrowth from each of the groups of mice of the in vivo experiment. FIG.11 is a photograph of the collection of excised tumors taken from micefrom each of groups and aligned alongside on a row corresponding to eachgroup.

The tumors excised from the untreated control group were considerablylarger at 60 days than those excised from the other two groups, showingcontinued aggressive growth of the pancreatic tumor cells. In contrast,the compound of Example 1 group exhibited considerable pancreatic tumorgrowth inhibition as compared to the untreated control group, andsubstantial effective tumor cell inhibition as compared to native formtriptolide. Referring now to FIG. 11 and FIG. 12, which show comparativetumor weight and tumor volume between the three groups respectively, thecontrol group tumors were considerably larger both in terms of weight(g) and volume (cm³) as compared to the triptolide and triptolideprodrug (compound 1) group tumor data.

Thus, when administered to a living mammal in vivo, the triptolideprodrug compound of the invention can effectively inhibit tumor growthand inhibitory effect effectiveness comparable to native non-prodrugform triptolide. As can be seen from the above data and figures, thepancreatic cancer tumor growth in the mice treated with nativetriptolide and triptolide prodrug (compound 1) for 60 days exhibitedsignificantly reduced tumor volume as compared with the untreatedcontrol group. Furthermore, it was significant that in both thetriptolide and triptolide prodrug group subjects, there was no apparentsignificant impact on body weight and no apparent signs of toxicity inthe subjects. Thus, the compounds of the invention can provide tumorinhibition and inhibit cancer cell growth and in particular pancreaticcancer cell growth. Additionally, the compounds of the invention couldalso provide the basis for effective treatment to inhibit pancreaticcancer with low toxic side-effects in living mammals.

EXAMPLE 9 Compound 1 Induced Tumor Regression in an Orthotopic MouseModel of Pancreatic Cancer (60 Day Dosing Study)

MIA PaCa-2 cells (1×10⁶) were suspended in matrigel and injected intothe tail of the pancreas of 4-6 week old female nude mice. Ten dayspost-cell implantation, mice were injected intraperitoneally withindicated concentrations of compound 1 (0.1, 0.15, 0.3, 0.6 or 0.9mg/kg) or 0.2 mg/kg Triptolide QD for 60 d. Control mice were injectedwith saline BID. Treatment was stopped after 60 d and mice were observedfor another 28 d before being sacrificed. Tumor samples, if any, wereharvested from these mice, and tumor weight and volume measured. Iftumor burden exceeded University of Minnesota animal care guidelines,mice were sacrificed at earlier time points and their tumors harvested.FIG. 13 illustrates the enhanced survival of mice treated with compound1 and triptolide versus vehicle. FIG. 14 illustrates the enhancedsurvival of mice treated with compound 1 versus vehicle. FIG. 15 showsthe decreased tumor burden, as measured by tumor volume or tumor weight,of compound 1 treated mice versus control mice.

EXAMPLE 10 Compound 1 Induced Tumor Regression in an Orthotopic MouseModel of Pancreatic Cancer (21 Day Dosing Study)

1×10⁶ Cells of S2013, a highly metastatic pancreatic cancer cell line,were suspended in matrigel and injected into the tail of the pancreas of4-6 week old female nude mice. Seven days post-cell implantation, micewere injected intraperitoneally with 0.42 mg/kg of compound 1 for 21 d.Treatment was stopped after 21 d, and mice sacrificed. Tumor samples, ifany, were harvested from these mice, and tumor weight and volumemeasured. If tumor burden exceeded University of Minnesota animal careguidelines, mice were sacrificed and their tumors harvested at anearlier time point. Control mice were injected with saline QD. FIG. 16shows the decreased tumor burden, as measured by tumor volume or tumorweight, of compound 1 treated mice versus control mice.

EXAMPLE 11 Compound 1 Induced Tumor Regression in a Subcutaneous MouseModel of Cholangiocarcinoma

SkChA-1cells (5×10⁵) were suspended in matrigel and injectedsubcutaneously into the left flank of 4-6 week old female nude mice.Seven days post-cell implantation, mice were injected intraperitoneallywith 0.3 mg/kg of compound 1 BID for 25 days. Treatment was stopped atthis point, and mice sacrificed. Tumor samples, if any, were harvestedfrom these mice, and tumor weight and volume measured. If tumor burdenexceeded University of Minnesota animal care guidelines, mice weresacrificed and their tumors harvested at an earlier time point. Controlmice were injected with saline BID. FIG. 17 shows the decreased tumorburden, as measured by tumor volume or tumor weight, of compound 1treated mice versus control mice.

EXAMPLE 12 Triptolide Induced Tumor Regression in a Orthotopic MouseModel of Neuroblastoma

Neuroblastoma N2 cells (1×10⁶) were suspended in matrigel and injectedinto the left retroperitoneal space of 4-6 week old A/J immunocompetentmice. Three days post-cell implantation, mice were injected with 0.4mg/kg of triptolide intraperitoneally for 21 days. Treatment was stoppedat this point, and mice sacrificed. Tumor samples, if any, wereharvested from these mice, and tumor weight and volume measured. Iftumor burden exceeded University of Minnesota animal care guidelines,mice were sacrificed and their tumors harvested at an earlier timepoint. Control mice were injected with DMSO for 21 days. FIG. 18 showsthe decreased tumor burden in the triptolide treated mice versus thecontrol mice as measured by tumor volume and tumor mass.

EXAMPLE 13 Triptolide Induced Cell Death and Capase-3 Activation inNeuroblastoma Cells

Neuroblastoma N2a and SKNSH cells were treated with triptolide,resulting in dose- and time-dependent cell killing in N2a, with morethan 50% of cells killed with 62.5 nM triptolide at 24 hours and nearly85% of cells killed with 250 nM triptolide at 48 hours (FIG. 19). Toconfirm the hypothesis that triptolide results in neuroblastoma celldeath via an apoptotic pathway, caspase-3 activity was measured as amarker of apoptosis. In both cell lines, increases in caspase activitywith higher doses of triptolide and longer duration of therapy.Triptolide treatment was associated with dose- and time-dependentincreases in caspase-3 activity levels (FIG. 20). These results suggestthat triptolide-mediated cell death occurs via the induction ofapoptosis.

EXAMPLE 14 The Following Illustrate Representative Pharmaceutical DosageForms, Containing a Compound of Formula I (‘Compound X’), forTherapeutic or Prophylactic Use in Humans

(i) Tablet 1 mg/tablet Compound X = 0.5 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 185 (ii) Tablet 2 mg/tablet Compound X = 1.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 481 iii) Capsule mg/capsule Compound X = 2.0Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch 120.0Magnesium stearate 3.0 468 (iv) Injection 1 (1 mg/ml) mg/ml Compound X =0.5 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to8.2-9) Water for injection q.s. ad 1 mL (v) Injection 1 (1 mg/ml) mg/mlCompound X = 1 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate0.7 Sodium chloride 4.5 1.0N Sodium hydroxide solution q.s. (pHadjustment to 8.2-9.0) Water for injection q.s. ad 1 mL (vi) Injection 2(10 mg/ml) mg/ml Compound X = 2 Monobasic sodium phosphate 0.3 Dibasicsodium phosphate 1.1 Polyethylene glycol 400 200.0 01N Sodium hydroxidesolution q.s. (pH adjustment to 8.2-9.0) Water for injection q.s. ad 1mLThe above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A compound of formula I:

wherein: each R¹ is independently H, (C₁-C₆)alkyl, aryl(C₁-C₆)alkyl-,(C₃-C₆)cycloalkyl or aryl; and each R² is independently H, (C₁-C₆)alkyl,aryl(C₁-C₆)alkyl-, (C₃-C₆)cycloalkyl or aryl; or R¹ and R² together withthe atom to which they are attached form a (C₃-C₇)cycloalkyl; whereinany alkyl or cycloalkyl of R¹ or R² may be optionally substituted withone or more groups selected from halo, (C₁-C₆)alkoxy and NR^(a)R^(b) andwherein any aryl of R¹ or R² may be optionally substituted with one ormore groups selected from halo, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,NR^(a)R^(b), nitro and cyano; R^(a) and R^(b) are each independentlyselected from H, (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl and aryl; or R^(a) andR^(b) together with the nitrogen to which they are attached form apyrrolidino, piperidino, piperazino, azetidino, morpholino, orthiomorpholino; n is 1, 2 or 3; and each X is H; or a salt thereof.
 2. Acompound of claim 1 which is a compound of formula Ia:

wherein X⁺ is a pharmaceutically acceptable organic cation or inorganiccation.
 3. The compound of claim 1 wherein R¹ is H or (C₁-C₆)alkyl. 4.The compound of claim 1 wherein R¹ is H.
 5. The compound of claim 1wherein R¹ is (C₁-C₆)alkyl.
 6. The compound of claim 1 wherein R¹ ismethyl or ethyl.
 7. The compound of claim 1 wherein R² is H or(C₁-C₆)alkyl.
 8. The compound of claim 1 wherein R² is H.
 9. Thecompound of claim 1 wherein each X⁺ is H.
 10. The compound of claim 1wherein each X⁺ is lithium, sodium, potassium, magnesium, calcium,barium, zinc or aluminium.
 11. The compound of claim 1 wherein each X⁺is of the formula HY⁺ wherein Y is ammonia, triethylamine, tromethamine,triethanolamine, ethylenediamine, glucamine, N-methylglucamine, glycine,lysine, ornithine, arginine, ethanolamine or choline.
 12. The compoundof claim 1 wherein X⁺ is selected from Li⁺, K⁺ and Na⁺.
 13. The compoundof claim 1 wherein each X⁺ is Na⁺.
 14. The compound of claim 1 which is14-O-phosphonooxymethyltriptolide disodium salt,14-O-phosphonooxyethyltriptolide disodium salt or14-O-phosphonooxypropyltriptolide disodium salt.
 15. The compound ofclaim 1 which is 14-O-phosphonooxy-methyltriptolide disodium salt.
 16. Apharmaceutical composition comprising a compound of formula I asdescribed in claim 1, or a pharmaceutically acceptable salt thereof, incombination with a pharmaceutically acceptable carrier.